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R R Stewart

Publications and source records attributed to R R Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1994

Membrane properties of microglial cells isolated from the leech central nervous system.

Membrane potentials and channel properties of microglial cells isolated from the leech central nervous system and maintained in culture on different substrates were investigated by using the patch-clamp technique. As expected, microglia on concanavalin A (con-A) were round and stationary, whereas those on extract of extracellular matrix (ECM) were spindle shaped and mobile. The mean membrane capacitance was 9 +/- 1 pF (s.e., n = 46), and the input resistance ranged from 0.135 G omega to 21 G omega with a mean of 4.2 +/- 1.6 G omega (n = 19). On-cell patches exhibited no single-channel activity. Voltage-dependent Na+, K+ and Ca2+ currents typical of neurons were absent. Currents evoked in response to voltage ramps from -100 mV to +100 mV or to steps of 4 s duration reversed in sign at or near 0 mV and exhibited single-channel activity of increasing amplitude for incrementally larger positive and negative voltage steps. No differences between the membrane properties of microglial cells on con-A and on ECM were evident. Currents were increased in fluid in which Na+ was substituted with K+, and were decreased when Na+ was substituted with N-methyl-D-glucamine. Varying external [Cl-] was without effect, as was addition to the fluid of 100 microM anthracene-9-carboxylate, a Cl- channel blocker. Together these characteristics indicated a cation channel. Bath application of 100 microM serotonin reversibly increased both inward and outward currents as well as single-channel activity. It is concluded that cultured microglial cells isolated from the adult leech have high membrane resistance and cation channel activity influenced by serotonin.

Animals↗
PubMed1991

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.

Aging↗
PubMed1991

Regulation of GABAB receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture.

The aim of these experiments has been to analyse the properties of receptors for the transmitter gamma-aminobutyric acid (GABA) in developing mammalian nervous system. Changes in responses of GABAB receptors have been measured after alterations of the chemical environment and the level of electrical activity. We have previously shown that when the central nervous system (CNS) of the new-born opossum, Monodelphis domestica, is cultured for three to five days in the presence of histidine, inhibition by baclofen, a GABAB agonist, disappears (Stewart et al. 1991). We have now investigated whether histidine acts indirectly by way of conversion to histamine. As with histidine, culture with 150 microM histamine for five days virtually abolished the inhibition by baclofen. The effects of histidine, as well as histamine, were blocked by mepyramine, a histamine H1-receptor antagonist, and by ranitidine, an H2-antagonist. Tetrodotoxin (TTX), which blocks all electrical activity, protected preparations from the action of histidine but not histamine. Our results suggest that histidine is converted to histamine, which reduces the efficacy of GABAB agonists. We conclude that, in the developing mammalian CNS, transmitter levels and electrical activity can selectively influence the properties of receptors.

Animals↗
PubMed1991

The intact central nervous system of the newborn opossum in long-term culture: fine structure and GABA-mediated inhibition of electrical activity.

1. The entire central nervous system (CNS) of the newly born, South American opossum (Monodelphis domestica) was isolated and maintained in basal medium, Eagle's (BME) with 0.2% foetal calf serum and antibiotics. Isolated CNS preparations remained electrically excitable for up to 10 days. The fine structure of the spinal cord was normal after 5 days in culture: axons, synapses, dendrites and glia were virtually unchanged. Signs of degeneration were evident only in dorsal areas of the spinal cord, which had been denervated by removal of the dorsal root ganglia during dissection. 2. Amino acid transmitters such as glycine, glutamate, N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA), applied to the bathing fluid, rapidly and reversibly inhibited synaptic transmission in cervical segments of the spinal cord. GABA (10-100 mumol l-1) produced a dose-dependent reduction in the magnitude of ventral root responses evoked by dorsal root stimulation. GABA also inhibited synaptically activated compound action potentials produced by spinal cord stimulation. Dose-response curves for GABA obtained in different preparations were highly reproducible. 3. Both GABAA and GABAB receptors were reversibly activated by selective agonists and inhibited by specific antagonists. The actions of GABA were potentiated by benzodiazepines, competitively antagonised by bicuculline (a selective GABAA antagonist) and mimicked by muscimol (a GABAA agonist). Baclofen (a specific GABAB agonist) also inhibited electrical activity and was competitively antagonised by the GABAB antagonist, CGP 35348. 4. After 5 days of culture in BME or minimal essential medium (MEM), GABA dose-response curves were unchanged from those observed immediately after removal of the CNS. The inhibitory potency of baclofen was also unaffected by culture in BME. By contrast, after 5 days of culture in MEM, baclofen no longer inhibited electrical activity. This difference between BME and MEM could be attributed to the higher content of L-histidine in MEM. Thus, addition of 150 mumol l-1 L-histidine to BME produced similar results to culture in MEM: the inhibitory action of baclofen was virtually abolished after 3-5 days. L-Histidine had no effect on freshly dissected preparations. Chronic application of L-histidine did not affect glycine or glutamate responses after 5 days. Addition of D-histidine or other amino acids, such as arginine, to BME did not abolish the responses to baclofen. 5. These results show that the isolated CNS of the newborn opossum survives well in long-term culture and that it provides a useful preparation to study receptor development and plasticity of an intact mammalian CNS in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1990

Reflexes, fictive respiration and cell division in the brain and spinal cord of the newborn opossum, Monodelphis domestica, isolated and maintained in vitro.

1. The entire central nervous system (CNS) was isolated from 1- to 4-day-old newborn South American opossums (Monodelphis domestica). At this stage the CNS has only an embryonic forebrain (two-layered) and no cerebellum and corresponds to a 14-day rat embryo. Its eyes, ears and hind-limbs are only at an early stage of formation. The isolated CNS preparations continue to develop and to produce electrical signals for up to 4 days in oxygenated Krebs' fluid at 23 degrees C. 2. The longitudinal axis of the CNS showed markedly different stages of development. More neuroblast cells were present in the proliferative zone in lumbosacral than in cervical or thoracic regions of the cord. 3. The progeny of dividing cells were labelled in isolated preparations by applying bromodeoxyuridine (BrdU) to the bathing solution for 2 h. Stained precursor cells were observed in CNS that had been left in Krebs' fluid for 4 days before applying BrdU and also in CNS that had been exposed to BrdU shortly after dissection and then left for 4 days. 4. Compound action potentials were evoked from the isolated CNS by stimulation with extracellular electrodes. Compound action potentials increased in amplitude with stronger stimulation and showed discrete peaks of conduction velocity. All electrical activity was eliminated reversibly by 0.1 mumol l-1 tetrodotoxin applied to the bathing solution. Block and recovery occurred with a half-time of approximately 5 min. High concentrations of magnesium (20 mmol l-1) reversibly blocked slower components of the volley. 5. Reflexes in cervical and thoracic segments of the spinal cord continued to function in isolated preparations. Stimulation of a dorsal root evoked bursts of impulses in the appropriate ventral root. Spontaneous and evoked activity in ventral roots was eliminated reversibly by 20 mmol l-1 magnesium. 6. In thoracic segments, spontaneous rhythmical bursts of action potentials were recorded. Burst activity was correlated with respiratory movements of the ribs in semi-intact preparations in which a few ribs and muscles were left attached to the isolated CNS. 7. At raised temperatures of 28 degrees C compared to 23 degrees C both spontaneous and evoked electrical activity were reversibly reduced. 8. Together these results show that the isolated CNS of the newborn opossum survives well in culture. The preparation offers advantages for pharmacological and physiological studies of spinal reflexes, for analysis of the mechanisms underlying rhythmical respiratory activity and for following the time course of CNS development in vitro.

Action Potentials↗
PubMed1989

Nucleotide sequence of the narL gene that is involved in global regulation of nitrate controlled respiratory genes of Escherichia coli.

The DNA sequence was determined for the narL gene of Escherichia coli. This gene is involved in global regulation of a number of nitrate controlled genes including frdABCD, tor, narGHJI, and adhE which are associated with bacterial respiration and fermentation. Comparison of the deduced amino acid sequence of narL to that of other bacterial genes revealed significant homologies to the phoB, ompR, and virG gene products based on the presence of similar protein domains. These DNA binding proteins are members of two-component regulatory systems. The similarities suggest that narL may also participate in such a two-component regulatory system and that the narR gene, which lies upstream of narL, may encode a second component required for nitrate control of gene regulation. In vitro protein synthesis experiments using a narL plasmid identified a putative NarL protein of 29 kDa in size consistent with the DNA sequence analysis. Primer extension experiments revealed the presence of two 5' termini for narL mRNA, and indicates that transcription may be complex.

Amino Acid Sequence↗
PubMed1989

Thyroid gland: US in patients with Hodgkin disease treated with radiation therapy in childhood.

The authors retrospectively assessed with sonography the prevalence of thyroid gland abnormalities in 30 patients who underwent radiation therapy for Hodgkin disease between 1962 and 1984. Doses ranged from 3,000 to 4,500 rad (3,000-4,500 cGy). Abnormalities were found in the sonograms of 24 patients and included unilateral (n = 6) or bilateral (n = 2) atrophy; multiple hypoechoic lesions smaller than 0.75 cm (n = 18); and dominant cystic (n = 2), solid (n = 3), or complex lesions (n = 4) larger than 0.75 cm. The risk of development of an abnormality increased as the time from irradiation increased and was comparable between patients who did and did not receive chemotherapy as part of the treatment regimen. Although the pathologic correlates of the various abnormalities seen on sonograms may differ, the findings indicate a need for long-term follow-up of patients who underwent cervical irradiation for Hodgkin disease.

Adolescent↗
PubMed1989

Na+, K+ and Ca2+ currents in identified leech neurones in culture.

1. Na+, K+ and Ca2+ currents have been measured by voltage-clamp in Retzius (R), anterior pagoda (AP) and sensory (pressure, touch and nociceptive) cells dissected from the central nervous system (CNS) of the leech. These cells maintain their distinctive membrane properties and action potential configurations in culture. Currents carried by the individual ions were analysed by the use of channel blockers and by their kinetics. Since the cells are isopotential they can be voltage-clamped effectively. 2. Depolarization, as expected, gave rise to an early inward Na+ current followed by a delayed outward K+ current. In Na+-free medium containing tetraethylammonium (TEA+), and in the presence of 4-aminopyridine (4-AP), inward Ca2+ currents were revealed that inactivated slowly and were blocked by Cd2+ and Mn2+. 3. Na+ and Ca2+ currents were similar in their characteristics in R. AP and sensory neurones. In contrast, K+ currents showed marked differences. Three principal K+ currents were identified. These differed in their time courses of activation and inactivation and in their responses to Ca2+ channel blockers. 4. K+ currents of the A-type (IA) activated and inactivated rapidly, were not affected by Ca2+ channel blockers and were eliminated by steady-state inactivation at holding potentials of -30 mV. A-type K+ currents were found in AP cells and as a minor component of the outward current in R cells. A Ca2+-activated K+ current (IC), that inactivated more slowly and was reduced by Ca2+ channel blockers, constituted the major outward current in R cells. The third K+ current resembled the delayed rectifier currents (IK1 and IK2) of squid axons with slow activation and inactivation kinetics. Such currents were found in R cells and in the sensory neurones (T, P and N). 5. The principal differences in membrane properties of identified leech neurones can be explained in terms of the numbers of Na+ channels and the distinctive kinetics of K+ channels in each type of cell.

4-Aminopyridine↗
PubMed1989

Presynaptic calcium currents and facilitation of serotonin release at synapses between cultured leech neurones.

1. The role of presynaptic Ca2+ entry in facilitation of transmitter release has been analysed by voltage-clamp measurements at synapses formed in culture by Retzius and P neurones isolated from the central nervous system (CNS) of the leech. The transmitter released by Retzius cells is serotonin. 2. Synaptic transmission persisted in solutions containing raised concentrations of divalent cations, reduced concentrations of Na+, and tetraethylammonium (TEA+) and 4-AP (to block K+ currents). Ca2+ and Sr2+ were more effective in promoting transmitter release than Ba2+, as assessed by the postsynaptic potentials in P cells. The degree and time course of facilitation in Ca2+- and Sr2+-containing solutions were similar to those observed for synapses bathed in normal L-15 medium. 3. Transmitter release depended upon the amplitude and the duration of presynaptic depolarization and inward Ca2+ current. Peak Ca2+ currents and postsynaptic potentials occurred with depolarizing steps to +15 mV. Frequent or prolonged pulses depressed the postsynaptic potentials. 4. Pairs of depolarizing pulses that caused facilitation were accompanied by identical inward Ca2+ currents. These results indicate that the mechanism responsible for facilitated serotonin release must occur following Ca2+ entry and that residual Ca2+ plays a role.

4-Aminopyridine↗
PubMed1987

Cell death during gangliogenesis in the leech: competition leading to the death of PMS neurons has both random and nonrandom components.

The posteromedial, serotonin-containing (PMS) neurons are found in the ventral aspect of certain anterior segmental ganglia of adult leeches. With one exception, these cells are unpaired in all the ganglia where they are found. During early embryogenesis in Hirudo medicinalis, however, a bilateral pair of PMS neurons appears and differentiates in each of the 21 segmental ganglia (SG1-SG21). Over the next several days, one of the pair dies in SG3-SG21. Examination of the PMS neuron in any one of these segments reveals that either the right or the left cell remains, with equal probability, suggesting that the elimination of one of the pair is a random process. When unpaired PMS neurons are examined in pairs of adjacent ganglia, however, the cells are from opposite sides in the majority of cases (approximately 88%). This observation implies that the death of a PMS neuron in 1 ganglion strongly biases which member of the pair of PMS neurons degenerates in adjacent ganglia. Detailed examination of the sequence of degeneration shows that it begins at several separate loci in the nerve cord. We propose that the mechanism responsible for the death of one of the pair of PMS neurons in a segmental ganglion is competition between these 2 cells for some trophic factor, but that the outcome of this competition is predetermined if one of the PMS neurons in an adjacent ganglion has already begun to degenerate.

Animals↗
PubMed1987

Cell death during gangliogenesis in the leech: bipolar cells appear and then degenerate in all ganglia.

The bipolar cells can be recognized very early during gangliogenesis in the leech central nervous system by their expression of antigens that are recognized by the monoclonal antibody Laz1-1. They are the first cells to express these antigens, which are later shared with a distinct set of other cells in the leech nervous system. Their processes extend several segments rostrally and caudally along the forming interganglionic connective nerves; they are first found in anterior segments and gradually appear in more posterior ones over the course of 2-3 d. At about the time bipolar cells appear in the neuromeres of the tail ganglion, those in the most anterior segmental ganglia begin to degenerate. Degeneration proceeds caudally over the next 3 d, until all bipolar cells have disappeared. Bipolar cells are, thus, members of that class of cells that exists for only a short period in neurogenesis and then presumably disappears once its functions are no longer required. These cells' morphology and appearance at the earliest stages in the formation of the interganglionic connective nerves are suggestive of a role in the establishment of these longitudinal pathways in the leech CNS.

Animals↗
PubMed1986

Segmental differentiation in the leech nervous system: the genesis of cell number in the segmental ganglia of Haemopis marmorata.

In hirudinid leeches, the segmental ganglia associated with the sexual organs contain several hundred more neurons than other midbody ganglia. To determine whether this difference arises by differential cell addition or by differential cell death, cell counts were made in several segmental ganglia during the course of embryonic and postembryonic development. The results show that all ganglia behave equally in early development. In each case, at least 10-20% more cells than will make up the adult complement of about 400 neurons is generated, and by about 20 days of embryonic development cell loss brings the number down to about 400 cells. By about 30 days, when animals emerge from their cocoons, additional cells have begun to appear in the sex ganglia. The number of extra cells continue to increase gradually over the next several months, until the adult number of 600-700 neurons is attained. These observations indicate that at least some segmental differences in the size of neuronal populations are due to differential cell proliferation and that these differences can arise quite late in the maturation of an animal.

Animals↗
PubMed1986

Segmental differentiation in the leech nervous system: specific phenotypic changes associated with ectopic targets.

The issue of whether interactions between central neurons and the targets they innervate are involved in establishing segment-specific phenotypic differences in the central nervous system was evaluated by studying the effect of naturally occurring ectopic targets on individual identified neurons of the leech Hirudo medicinalis. The specimens studied were found to have additional sex organs located in segments adjacent to those containing the normal structures. We report our observations on neuronal morphology, the presence or absence of specific neurons, and total ganglionic cell number and discuss some of the possible ways in which the observed changes might have arisen.

Animals↗
PubMed1985

Expression of surface antigens recognized by the monoclonal antibody lan 3-2 during embryonic development in the leech.

The monoclonal antibody lan 3-2 was used as a marker for the developmental expression of surface antigens which are specific for the two pairs of nociceptive neurons and a subset of axons in the adult CNS of the leech. In Haemopis embryos labeling of both nerve fibers and cell bodies with the antibody appears as expected for a metameric animal in a rostrocaudal temporal gradient from about day 5-6. Surprisingly, all central cell bodies are stained by the antibody in early development. However, later in embryogenesis around day 17 the staining intensity of most cells decreases except for the nociceptive cells, which remain antibody-positive, and the adult staining pattern gradually emerges. In addition to describing the central staining pattern, we show that specific peripheral neurons associated with the segmental sensilla also are antibody-positive during development. The distribution and developmental expression of the lan 3-2-positive antigens are compared between two phylogenetically different species of leeches and the diversity of the staining pattern of the monoclonal antibody is discussed.

Animals↗
PubMed1985

The embryonic development of peripheral neurons in the body wall of the leech Haemopis marmorata.

The appearance of peripheral neurons within the skin during embryonic development of the leech is described. These neurons were labeled using a monoclonal antibody, Lan3-6, which recognizes antigens in both the cell body and the axons of these cells. Within the 5 annuli that are found in each midbody segment, peripheral neurons first label in the middle and last in the most anterior and posterior ones. In each annulus, the number of cells labeled is initially 4 and increases as development proceeds. By the end of embryogenesis, all annuli show approximately equal numbers of Lan3-6 labeled neurons. The development of peripheral neurons in the skin of the rear sucker is also described.

Animals↗
PubMed1983

Metorchis conjunctus (Cobbold, 1860) infection in wolves (Canis lupus), with pancreatic involvement in two animals.

The trematode Metorchis conjunctus (Cobbold, 1860) was found in seven of 211 wolves from Saskatchewan which were examined between 1976 and 1983. The parasite caused cholangiohepatitis with periductular fibrosis in the liver of all the wolves, and chronic inflammation and fibrosis of the pancreas in two animals. The wolves with pancreatic involvement were emaciated. Five of the seven infected wolves were from one local area, and three of these were from a pack known to consume fish.

Animals↗